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. 1986 Sep 1;103(3):947–956. doi: 10.1083/jcb.103.3.947

Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

PMCID: PMC2114312  PMID: 3091608

Abstract

Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP- sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility.

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Selected References

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  1. Allen R. D., Allen N. S. Video-enhanced microscopy with a computer frame memory. J Microsc. 1983 Jan;129(Pt 1):3–17. doi: 10.1111/j.1365-2818.1983.tb04157.x. [DOI] [PubMed] [Google Scholar]
  2. Allen R. D., Weiss D. G., Hayden J. H., Brown D. T., Fujiwake H., Simpson M. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol. 1985 May;100(5):1736–1752. doi: 10.1083/jcb.100.5.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell C. W., Fraser C., Sale W. S., Tang W. J., Gibbons I. R. Preparation and purification of dynein. Methods Cell Biol. 1982;24:373–397. doi: 10.1016/s0091-679x(08)60666-4. [DOI] [PubMed] [Google Scholar]
  4. Brady S. T. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature. 1985 Sep 5;317(6032):73–75. doi: 10.1038/317073a0. [DOI] [PubMed] [Google Scholar]
  5. Brady S. T., Lasek R. J., Allen R. D. Fast axonal transport in extruded axoplasm from squid giant axon. Science. 1982 Dec 10;218(4577):1129–1131. doi: 10.1126/science.6183745. [DOI] [PubMed] [Google Scholar]
  6. Brady S. T., Lasek R. J., Allen R. D. Video microscopy of fast axonal transport in extruded axoplasm: a new model for study of molecular mechanisms. Cell Motil. 1985;5(2):81–101. doi: 10.1002/cm.970050203. [DOI] [PubMed] [Google Scholar]
  7. Brady S. T., Lasek R. J., Allen R. D., Yin H. L., Stossel T. P. Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments. Nature. 1984 Jul 5;310(5972):56–58. doi: 10.1038/310056a0. [DOI] [PubMed] [Google Scholar]
  8. Brady S. T., Lasek R. J. Axonal transport: a cell-biological method for studying proteins that associate with the cytoskeleton. Methods Cell Biol. 1982;25(Pt B):365–398. doi: 10.1016/s0091-679x(08)61434-x. [DOI] [PubMed] [Google Scholar]
  9. Caceres A., Binder L. I., Payne M. R., Bender P., Rebhun L., Steward O. Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J Neurosci. 1984 Feb;4(2):394–410. doi: 10.1523/JNEUROSCI.04-02-00394.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Camilli P., Miller P. E., Navone F., Theurkauf W. E., Vallee R. B. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience. 1984 Apr;11(4):817–846. [PubMed] [Google Scholar]
  11. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  12. Gibbons I. R., Rowe A. J. Dynein: A Protein with Adenosine Triphosphatase Activity from Cilia. Science. 1965 Jul 23;149(3682):424–426. doi: 10.1126/science.149.3682.424. [DOI] [PubMed] [Google Scholar]
  13. Gilbert S. P., Allen R. D., Sloboda R. D. Translocation of vesicles from squid axoplasm on flagellar microtubules. Nature. 1985 May 16;315(6016):245–248. doi: 10.1038/315245a0. [DOI] [PubMed] [Google Scholar]
  14. Gilbert S. P., Sloboda R. D. Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei. J Cell Biol. 1984 Aug;99(2):445–452. doi: 10.1083/jcb.99.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldberg D. J. Microinjection into an identified axon to study the mechanism of fast axonal transport. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4818–4822. doi: 10.1073/pnas.79.15.4818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grafstein B., Forman D. S. Intracellular transport in neurons. Physiol Rev. 1980 Oct;60(4):1167–1283. doi: 10.1152/physrev.1980.60.4.1167. [DOI] [PubMed] [Google Scholar]
  17. Haley B. E., Hoffman J. F. Interactions of a photo-affinity ATP analog with cation-stimulated adenosine triphosphatases of human red cell membranes. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3367–3371. doi: 10.1073/pnas.71.9.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hoppe J., Freist W. Localization of the high-affinity ATP site in adenosine-3':5'-monophosphate-dependent protein kinase type I. Photoaffinity labelling studies with 8-azidoadenosine 5'-triphosphate. Eur J Biochem. 1979 Jan 2;93(1):141–146. doi: 10.1111/j.1432-1033.1979.tb12804.x. [DOI] [PubMed] [Google Scholar]
  19. Huber G., Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984 Jan;4(1):151–160. doi: 10.1523/JNEUROSCI.04-01-00151.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Isenberg G., Schubert P., Kreutzberg G. W. Experimental approach to test the role of actin in axonal transport. Brain Res. 1980 Aug 4;194(2):588–593. doi: 10.1016/0006-8993(80)91247-0. [DOI] [PubMed] [Google Scholar]
  21. Jacobs M., Smith H., Taylor E. W. Tublin: nucleotide binding and enzymic activity. J Mol Biol. 1974 Nov 5;89(3):455–468. doi: 10.1016/0022-2836(74)90475-6. [DOI] [PubMed] [Google Scholar]
  22. Kendrick-Jones J., Lehman W., Szent-Györgyi A. G. Regulation in molluscan muscles. J Mol Biol. 1970 Dec 14;54(2):313–326. doi: 10.1016/0022-2836(70)90432-8. [DOI] [PubMed] [Google Scholar]
  23. Kerlavage A. R., Taylor S. S. Covalent modification of an adenosine 3':5'-monophosphate binding site of the regulatory subunit of cAMP-dependent protein kinase II with 8-azidoadenosine 3':5'-monophosphate. Identification of a single modified tyrosine residue. J Biol Chem. 1980 Sep 25;255(18):8483–8488. [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lasek R. J., Brady S. T. Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP. Nature. 1985 Aug 15;316(6029):645–647. doi: 10.1038/316645a0. [DOI] [PubMed] [Google Scholar]
  27. Lasek R. J., Krishnan N., Kaiserman-Abramof I. R. Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons. J Cell Biol. 1979 Aug;82(2):336–346. doi: 10.1083/jcb.82.2.336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. MARTONOSI A., GOUVEA M. A., GERGERLY J. Studies on actin. I. The interaction of C14-labeled adenine nucleotides with actin. J Biol Chem. 1960 Jun;235:1700–1703. [PubMed] [Google Scholar]
  29. Maruta H., Korn E. D. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins. J Biol Chem. 1981 Jan 10;256(1):499–502. [PubMed] [Google Scholar]
  30. Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miller R. H., Lasek R. J. Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm. J Cell Biol. 1985 Dec;101(6):2181–2193. doi: 10.1083/jcb.101.6.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Morris J. R., Lasek R. J. Stable polymers of the axonal cytoskeleton: the axoplasmic ghost. J Cell Biol. 1982 Jan;92(1):192–198. doi: 10.1083/jcb.92.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  34. Murphy D. B., Wallis K. T., Hiebsch R. R. Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria. J Cell Biol. 1983 May;96(5):1306–1315. doi: 10.1083/jcb.96.5.1306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Neidl C., Engel J. Exchange of ADP, ATP and 1: N6-ethenoadenosine 5'-triphosphate at G-actin. Equilibrium and kinetics. Eur J Biochem. 1979 Nov 1;101(1):163–169. doi: 10.1111/j.1432-1033.1979.tb04228.x. [DOI] [PubMed] [Google Scholar]
  36. Pant H. C., Gallant P. E., Gainer H. Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon. J Biol Chem. 1986 Feb 25;261(6):2968–2977. [PubMed] [Google Scholar]
  37. Pant H. C., Shecket G., Gainer H., Lasek R. J. Neurofilament protein is phosphorylated in the squid giant axon. J Cell Biol. 1978 Aug;78(2):R23–R27. doi: 10.1083/jcb.78.2.r23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Papasozomenos S. C., Binder L. I., Bender P. K., Payne M. R. Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons. J Cell Biol. 1985 Jan;100(1):74–85. doi: 10.1083/jcb.100.1.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Papasozomenos S. C., Yoon M., Crane R., Autilio-Gambetti L., Gambetti P. Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study. J Cell Biol. 1982 Nov;95(2 Pt 1):672–675. doi: 10.1083/jcb.95.2.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pfister K. K., Haley B. E., Witman G. B. Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP. J Biol Chem. 1985 Oct 15;260(23):12844–12850. [PubMed] [Google Scholar]
  41. Pfister K. K., Haley B. E., Witman G. B. The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein. J Biol Chem. 1984 Jul 10;259(13):8499–8504. [PubMed] [Google Scholar]
  42. Potter R. L., Haley B. E. Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: techniques and applications. Methods Enzymol. 1983;91:613–633. doi: 10.1016/s0076-6879(83)91054-6. [DOI] [PubMed] [Google Scholar]
  43. Pratt M. M., Hisanaga S., Begg D. A. An improved purification method for cytoplasmic dynein. J Cell Biochem. 1984;26(1):19–33. doi: 10.1002/jcb.240260103. [DOI] [PubMed] [Google Scholar]
  44. Pratt M. M. Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity. J Cell Biol. 1986 Sep;103(3):957–968. doi: 10.1083/jcb.103.3.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  46. Scheurich P., Schäfer H. J., Dose K. 8-Azido-adenosine 5'-triphosphate as a photoaffinity label for bacterial F1 ATPase. Eur J Biochem. 1978 Jul 17;88(1):253–257. doi: 10.1111/j.1432-1033.1978.tb12445.x. [DOI] [PubMed] [Google Scholar]
  47. Schnapp B. J., Reese T. S. Cytoplasmic structure in rapid-frozen axons. J Cell Biol. 1982 Sep;94(3):667–669. doi: 10.1083/jcb.94.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schnapp B. J., Vale R. D., Sheetz M. P., Reese T. S. Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell. 1985 Feb;40(2):455–462. doi: 10.1016/0092-8674(85)90160-6. [DOI] [PubMed] [Google Scholar]
  49. Scholey J. M., Porter M. E., Grissom P. M., McIntosh J. R. Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle. Nature. 1985 Dec 5;318(6045):483–486. doi: 10.1038/318483a0. [DOI] [PubMed] [Google Scholar]
  50. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  51. Sloboda R. D., Rosenbaum J. L. Decoration and stabilization of intact, smooth-walled microtubules with microtubule-associated proteins. Biochemistry. 1979 Jan 9;18(1):48–55. doi: 10.1021/bi00568a008. [DOI] [PubMed] [Google Scholar]
  52. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  54. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Vale R. D., Schnapp B. J., Mitchison T., Steuer E., Reese T. S., Sheetz M. P. Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell. 1985 Dec;43(3 Pt 2):623–632. doi: 10.1016/0092-8674(85)90234-x. [DOI] [PubMed] [Google Scholar]
  57. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon. Cell. 1985 Mar;40(3):559–569. doi: 10.1016/0092-8674(85)90204-1. [DOI] [PubMed] [Google Scholar]
  58. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wagenvoord R. J., Van der Kraan I., Kemp A. Specific photolabelling of beef-heart mitochondrial ATPase by 8-azido-ATP. Biochim Biophys Acta. 1977 Apr 11;460(1):17–24. doi: 10.1016/0005-2728(77)90147-5. [DOI] [PubMed] [Google Scholar]
  60. Wiche G., Briones E., Hirt H., Krepler R., Artlieb U., Denk H. Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A). EMBO J. 1983;2(11):1915–1920. doi: 10.1002/j.1460-2075.1983.tb01679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yue V. T., Schimmel P. R. Direct and specific photochemical cross-linking of adenosine 5'-triphosphate to an aminoacyl-tRNA synthetase. Biochemistry. 1977 Oct 18;16(21):4678–4684. doi: 10.1021/bi00640a023. [DOI] [PubMed] [Google Scholar]
  62. Zabrecky J. R., Cole R. D. ATP-induced aggregates of tubulin rings. J Biol Chem. 1980 Dec 25;255(24):11981–11985. [PubMed] [Google Scholar]
  63. Zabrecky J. R., Cole R. D. Effect of ATP on the kinetics of microtubule assembly. J Biol Chem. 1982 Apr 25;257(8):4633–4638. [PubMed] [Google Scholar]

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